Gas6 fusion protein, preparation method therefor and use thereof

Abstract

Provided are a GAS6 fusion protein, a preparation method therefor and the use thereof. The provided fusion protein contains an scFv capable of specifically recognizing tumor cell antigens, and can disguise tumor cells as apoptotic cells and enable phagocytosis of tumor cells by phagocytes by means of interaction with TAM RTK. The provided GAS6 fusion protein is applicable for phagocytosis of various tumor cells, showing strong broad-spectrum properties.

Description

GAS6 fusion protein, its preparation method and application Technical Field

[0001] This application belongs to the field of biological immunology, specifically relating to a GAS6 fusion protein and its preparation method and application. Background Technology

[0002] The TYRO 3, AXL, and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in a variety of cancers and have been identified as promising therapeutic targets based on their different functions in cancer cells and pro-tumor immune cells.

[0003] TAM RTKs are expressed by epithelial cells, platelets, and various immune cells, and play important roles in tissue homeostasis and immune regulation. Three TAM receptor tyrosine kinases (RTKs)—TYR03, AXL, and MERTK (TAM)—share a common structure, including two immunoglobulin-like (Ig-like) and two fibronectin III (FNIII) domains in their extracellular N-terminal region, a single-pass transmembrane domain, and a cytoplasmic C-terminal region containing the kinase domain. TAM RTKs induce homodimerization, autophosphorylation, and kinase activation through ligand binding, thereby altering the control of subsequent downstream signaling and participating in cancer development.

[0004] Currently, the two most studied TAM ligands are growth arrest-specific protein 6 (GAS6) and the PROS1 secreted protein. TAM RTKs bridge the interaction between phosphatidylserine (PtdSer) on the apoptotic cell membrane and the TAM receptor via soluble mediators GAS6 or PROS1. The unique PtdSer-GAS6-TAM complex, the maximally activated mode of the RTK family, has been shown to be crucial in the sustained clearance of apoptotic cells and debris from various tissues.

[0005] Endocytosis is the process by which phagocytes clear apoptotic cells. The tyrosine kinase family of TAM (TYRO3, AXL and MERTK) receptors of phagocytes recognize the "Eat-me" signal and initiate endocytosis after interacting with PtdSer through GAS6 or PROS1, thereby achieving the effect of phagocytosis and clearing apoptotic cells and debris.

[0006] In cancer cells, TAM RTK activation of the signaling pathway promotes cancer cell survival, metastasis, and resistance to various chemotherapeutic agents and targeted therapies. Simultaneously, TAM RTK also plays a role in innate immune cells; TAM RTK activation promotes an immunosuppressive phenotype, producing anti-inflammatory cytokines and chemokines, inhibiting pro-inflammatory mediators, and reducing antigen presentation. Furthermore, TAM RTK can recruit immunosuppressive myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) and activate immune checkpoints.

[0007] Therefore, TAM antagonists hold great promise as an immune-mediated therapy for inhibiting a single target or in combination with other cancer therapies. Many TYRO3, AXL, and / or MERTK (TAM)-targeting drugs are being developed as cancer therapies, some of which have progressed to clinical testing, including selective small molecule tyrosine kinase inhibitors (TKIs), decoy receptor fusion proteins, antagonistic monoclonal antibodies, antibody-drug conjugates, and AXL-directed chimeric antigen receptor (CAR) T cells. There are also several emerging preclinical drugs, including inactive ligands and DNA aptamers that block the binding of TAM receptor tyrosine kinase (RTK) to endogenous ligands, small interfering RNA (siRNA) delivered by nanoparticles that silence TAM gene expression at the mRNA level, and heterobifunctional protein degraders that induce TAM RTK ubiquitination and subsequent proteasome degradation.

[0008] The closest existing technology for GAS6-mediated phagocytosis mainly involves the invention and application of chimeric antigen receptor (CAR) immune cells constructed based on GAS6. This provides a GAS6-modified chimeric antigen receptor (CAR) whose extracellular binding domain specifically targets the GAS6 receptor. The aim is to give CAR immune cells strong specificity and target affinity, thus resulting in strong cytotoxicity against target cells and high safety.

[0009] However, current traditional phagocytosis-related technologies based on GAS6 have the problem of limited targeting and lack of broad-spectrum application.

[0010] Summary of the Invention

[0011] Based on this, according to various embodiments of this application, a GAS6 fusion protein is provided, the technical solution of which is as follows:

[0012] This application provides a GAS6 fusion protein, which includes, from the N-terminus to the C-terminus, an antigen-specific binding protein, a linker, and GAS6c.

[0013] The amino acid sequence of GAS6c is shown in SEQ ID NO.1.

[0014] In one embodiment, the C-terminus of the GAS6 fusion protein further includes a fragment encoded by the EPM gene as shown in SEQ ID NO.2.

[0015] In one embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0016] In one embodiment, the N-terminus of the GAS6 fusion protein further includes a signal peptide as shown in SEQ ID NO.11;

[0017] The signal peptide includes one or more of GAS6, CSF2RA, and hCD8A.

[0018] In one embodiment, the linker includes one or more flexible linkers.

[0019] In one embodiment, the amino acid sequence of the linker is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0020] In one embodiment, the antigen-specific binding protein includes one or more of single-chain antibodies, ligands, and antibodies modified to bind.

[0021] In one embodiment, the amino acid sequence is as shown in SEQ ID NO.7 or SEQ ID NO.8.

[0022] In one embodiment, the amino acid sequence is as shown in SEQ ID NO.9 or SEQ ID NO.10.

[0023] In one embodiment, the GAS6 fusion protein is delivered via MSC.

[0024] This application provides a nucleic acid molecule comprising encoding the aforementioned GAS6 fusion protein.

[0025] This application provides a recombinant vector comprising the aforementioned nucleic acid molecules;

[0026] The recombinant vector is either a eukaryotic expression vector or a prokaryotic expression vector.

[0027] This application provides a recombinant cell that expresses the above-mentioned GAS6 fusion protein, or contains the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector;

[0028] The recombinant cells are eukaryotic or prokaryotic cells.

[0029] This application also provides a method for preparing a GAS6 fusion protein, comprising:

[0030] The recombinant cells described above were cultured, and the GAS6 fusion protein was isolated from the resulting culture.

[0031] This application also provides a pharmaceutical composition comprising the GAS6 fusion protein as described above, and pharmaceutically acceptable excipients.

[0032] In one embodiment, the pharmaceutical composition further includes immune cells.

[0033] In one embodiment, the immune cells include one or more of monocytes or macrophages, dendritic cells, natural killer cells, or natural killer T cells;

[0034] The macrophages include M1 type macrophages or M2 type macrophages.

[0035] This application, in another aspect, provides a method for treating a subject's disease, comprising:

[0036] Administer a therapeutically effective amount of the pharmaceutical composition to the subject.

[0037] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 is a schematic diagram of the GAS6 fusion protein modification;

[0040] Figure 2 shows the vector map of the pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c plasmid construction.

[0041] Figure 3 shows the vector map of the plasmid construction of pCDH-SFFV-copGFP-EF1a-myc-TRP1-TA99 scFv-GAS6c;

[0042] Figure 4 shows the results of human primary macrophages phagocytosis of N87 in vitro mediated by the HER2-GAS6c fusion protein.

[0043] Figure 5 shows the results of in vitro phagocytosis of N87-Fluc by RAW264.7 after adenovirus infection mediated by HER2-GAS6c fusion protein;

[0044] Figure 6 shows the expression of the TRP1-TA99-scFv-GAS6c fusion protein in cells and supernatant after transient transfection;

[0045] Figure 7 shows the results of RAW264.7 phagocytosis of B16 mediated by the TRP1-TA99-GAS6c fusion protein in vitro. Detailed Implementation

[0046] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0047] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0048] the term

[0049] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0052] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0053] This application provides a GAS6 fusion protein, which includes a signal peptide, an antigen-specific binding protein, a linker, and GAS6c sequentially from the N-terminus to the C-terminus, wherein the amino acid sequence of GAS6c is shown in SEQ ID NO.1.

[0054] The amino acid sequence of human GAS6c is shown in SEQ ID NO.1:

[0055] In one specific example, it also includes an EPM fragment as shown in SEQ ID NO.2.

[0056] The amino acid sequence of EPM is shown in SEQ ID NO.2:

[0057] ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY

[0058] It is understood that the scFv described in this application can be one, two or more (e.g., three or four) of any antibody or antigen-binding fragment.

[0059] Alternatively, the sequence of scFv is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0060] The amino acid sequence of human HER2-scFv is shown in SEQ ID NO.3:

[0061] The amino acid sequence of TRP1-TA99-scFv is shown in SEQ ID NO.4:

[0062] In a specific example, the linker includes, but is not limited to, one or more flexible linkers.

[0063] Alternatively, the linker sequence may be as shown in SEQ ID NO.5 or SEQ ID NO.6.

[0064] The amino acid sequence of the G4S linker is shown in SEQ ID NO.5: GGGGSGGGGSGGGGS

[0065] The amino acid sequence of the G3S linker is shown in SEQ ID NO.6: GGGS

[0066] Optionally, the amino acid sequence of the GAS6 fusion protein is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0067] The amino acid sequence of HER2-scFv-G4S-GAS6c-EPM is shown in SEQ ID NO.7:

[0068] The amino acid sequence of HER2-scFv-G4S-GAS6c is shown in SEQ ID NO.8:

[0069] Further, optionally, the amino acid sequence of the GAS6 fusion protein targeting TRP1 is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0070] The amino acid sequence of TRP1-TA99-scFv-GAS6c-EPM is shown in SEQ ID NO.9:

[0071] The amino acid sequence of TRP1-TA99-scFv-GAS6c is shown in SEQ ID NO.10.

[0072] Optionally, the amino acid sequence of the signal peptide of GAS6 is shown in SEQ ID NO.11:

[0073] MAPSLSPGPAALRRAPQLLLLLLAAECALA

[0074] In one specific example, the GAS6 fusion protein is delivered via, but is not limited to, MSC delivery.

[0075] This application provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned GAS6 fusion protein. The full-length nucleotide sequence or a partial fragment thereof capable of specifically recognizing a target cell antigen (scFv) can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis.

[0076] More specifically, the target cells include, but are not limited to, tumor cells, apoptotic cells, and senescent cells.

[0077] This application also provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0078] Optionally, the recombinant vector is a eukaryotic expression vector. This application does not specifically limit the type of eukaryotic expression vector, which may include, but is not limited to, HEK293FT cells.

[0079] This application also provides a recombinant cell comprising the aforementioned nucleic acid molecules or the aforementioned vector.

[0080] Optionally, the recombinant cells are eukaryotic cells or prokaryotic cells.

[0081] This application also provides a method for preparing a GAS6 fusion protein, comprising:

[0082] The recombinant cells described above were cultured, and the GAS6 fusion protein was isolated from the resulting culture.

[0083] This application also provides a pharmaceutical composition comprising the GAS6 fusion protein as described above and pharmaceutically acceptable excipients.

[0084] It is understood that the pharmaceutical composition may include, but is not limited to:

[0085] (1) GAS6 fusion protein + commonly used excipients;

[0086] (2) GAS6 fusion protein + macrophages + common excipients;

[0087] (3) GAS6 fusion protein + macrophages + other immune cells + commonly used excipients;

[0088] (4) GAS6 fusion protein + immune cells that phagocytose apoptotic cells + commonly used excipients;

[0089] (5) GAS6 fusion protein + immune cells + commonly used excipients.

[0090] This application does not impose any special restrictions on the types of excipients. Appropriate pharmaceutical excipients can be selected and the dosage of excipients can be adjusted according to the dosage form required in clinical practice.

[0091] Furthermore, the pharmaceutical compositions disclosed in this application can be used to treat tumors, with indications including: esophageal tumors such as squamous cell carcinoma, adenocarcinoma, lymphoma, neuroendocrine tumors, and sarcomas; gastric tumors such as leiomyomas, hemangiomas, lipomas, as well as gastric stromal tumors, gastric sarcomas, and gastric cancer; intestinal tumors such as colorectal polyps, colon cancer, rectal cancer, colonic carcinoids, and rectal cancer; liver tumors including focal nodular hyperplasia of the liver, hepatic hemangioma, hepatic cysts, hepatic adenomas, cirrhosis, and liver cancer; skin tumors such as sebaceous cysts, sebaceous nevi, syringomas, keloids, as well as fibrosarcomas, basal cell carcinoma, squamous cell carcinoma, and eczematous carcinoma; and also hematologic malignancies, nervous system tumors, urinary system tumors, and head and neck malignancies.

[0092] Furthermore, the pharmaceutical composition disclosed in this application can also be used to treat chronic inflammatory diseases or inflammation-related disease types, i.e., the indications further include: the more common ones include chronic pharyngitis, chronic gastritis, chronic pancreatitis, etc.

[0093] Furthermore, the inflammatory diseases mentioned therein include, but are not limited to, infectious and non-infectious inflammation, as well as degenerative, exudative, proliferative, and specific inflammation.

[0094] Furthermore, the inflammatory diseases include abnormal activation of inflammatory pathways in the absence of antigenic stimulation of the autoimmune system, including Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis, primary biliary cirrhosis, etc., as well as inflammatory diseases caused by infections such as bacteria, viruses, fungi, mycoplasma, chlamydia, rickettsia, etc.

[0095] In this application, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0096] In this application, "pharmaceuticalally acceptable excipient" means pharmaceutically acceptable material, composition or mediator, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials.

[0097] As used herein, the phrase “pharmaceuticalally acceptable excipient” includes buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier must be “pharmaceuticalally acceptable” in the sense of being compatible with other components in the formulation and harmless to the patient.

[0098] Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn... Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0099] In this application, "excipients" include, but are not limited to, mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, fructooligosaccharides, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipids, kaolin, talc, calcium stearate, and magnesium stearate.

[0100] This application also provides a method for TAM receptor phagocytosis mediated by GAS6 fusion protein, comprising: obtaining the GAS6 fusion protein as described above, synthesizing a plasmid vector encoding the GAS6 fusion protein, transfecting cells, isolating a culture containing the GAS6 fusion protein, and co-culturing tumor cells and immune cells under these conditions, thereby enabling GAS6-mediated TAM receptor-activated phagocytosis.

[0101] Optionally, immune cells include, but are not limited to, one or more of the following: monocytes, macrophages, dendritic cells, natural killer cells, or natural killer T cells.

[0102] Immune cells, commonly known as white blood cells, include lymphocytes and various phagocytes, and specifically refer to lymphocytes that can recognize antigens and produce specific immune responses. Lymphocytes are the basic components of the immune system and are widely distributed in the body. Primarily, T lymphocytes and B lymphocytes are activated by antigen stimulation, proliferate, and produce specific immune responses. In addition to T lymphocytes and B lymphocytes, there are also K lymphocytes and NK lymphocytes, making a total of four types. T lymphocytes are a multifunctional cell group. Besides lymphocytes, cells involved in the immune response include plasma cells, granulocytes, mast cells, antigen-presenting cells, and cells of the mononuclear phagocyte system.

[0103] Further, optionally, the macrophages include M1 macrophages or M2 macrophages.

[0104] This application also provides a method for treating a disease in a subject, comprising the steps of administering a therapeutically effective amount of the above-mentioned GAS6 fusion protein therapeutic agent to the subject.

[0105] It is understandable that the acquisition of GAS6 fusion proteins is not limited to synthesis within cells via DNA, cDNA, or mRNA, but can also occur either within the cell or secreted outside the cell.

[0106] This application modifies GAS6 by replacing the Gla domain (which recognizes PtdSer on the surface of apoptotic cells) in the human GAS6 sequence with a single-chain variable fragment (scFv) that recognizes tumor cell antigens, and by truncating the LG domain of GAS6 (which binds to the TAM receptor to induce receptor dimerization and activation), resulting in the GAS6 fusion protein, scFv-GAS6c. The GAS6 fusion protein provided in this application enables GAS6-TAM pathway-mediated phagocytosis via a bridging mechanism.

[0107] The scFv component of the GAS6 fusion protein specifically recognizes tumor cell antigens, while GAS6c in the GAS6 fusion protein interacts with TAM RTK, enabling phagocytes to engulf tumor cells. Therefore, the GAS6 fusion protein of this application can specifically recognize tumor cell antigens and is suitable for engulfing a variety of tumor cells, exhibiting broad-spectrum efficacy. For example, the GAS6 fusion protein drug of this application can be used for diseases such as osteoarthritis, Crohn's disease, Parkinson's disease, Alzheimer's disease, lupus erythematosus, and age-related diseases.

[0108] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0109] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0110] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0111] Example 1: scFv-GAS6c protein

[0112] This application replaces the Gla domain in the human GAS6 sequence with a single-chain variable fragment (scFv) that recognizes tumor cell antigens, and extracts the LG domain of GAS6, amino acids 298-678, to generate scFv-GAS6c. A schematic diagram of the specific modification is shown in Figure 1.

[0113] Gene fragments containing a myc tag, GAS6 signal peptide, anti-HER2 or other antigen single-stranded variable region (scFv), linker, GAS6c, and EPM are sequentially linked. The corresponding nucleotide sequences are obtained through artificial synthesis or PCR and inserted into the lentiviral pCDH-SFFV-copGFP-EF1a-puro vector or adenovirus vector. The recombinant plasmid is then identified by enzyme digestion.

[0114] Next, the plasmid was extracted using an endotoxin-free small-volume extraction kit from Omega. The extracted plasmid was then transiently transfected into HEK-293T cells using jetPRIME, and the supernatant was collected as conditioned medium. It should be noted that the plasmid extraction and transfection methods used in this application are only a specific embodiment, and the extraction methods and transfection protocols include, but are not limited to, the methods described above. To verify the mediated killing effect of the GAS6 fusion protein disclosed in this application, human primary macrophages were co-cultured in conditioned medium to perform in vitro phagocytosis or killing experiments on N87 or N87-Fluc, or other tumor target cells. The scFv-GAS6c mediated the phagocytic killing of N87 or N87-Fluc, or other tumor target cells by human primary macrophages.

[0115] Furthermore, to verify the effect of the GAS6 fusion protein disclosed in this application on the mediated killing effect of macrophages with different phenotypes, human primary macrophages were stimulated to the M1 phenotype using IFNγ or LPS, or human primary macrophages were stimulated to the M2 phenotype using IL4, and then co-cultured in conditioned medium to perform in vitro phagocytosis or killing of N87 or N87-Fluc, or other tumor target cells. The scFv-GAS6c mediated the phagocytosis and killing of human primary macrophages or other monocytes targeting N87 or N87-Fluc, or other tumor target cells. The GAS6 fusion protein disclosed in this application achieves mediated killing of macrophages with different phenotypes independent of macrophage phenotype.

[0116] Furthermore, to verify the effect of the GAS6 fusion protein disclosed in this application on the mediated killing effect of different types of effector cells, in vitro phagocytosis or killing of N87 or N87-Fluc, or other tumor target cells was performed by co-culturing monocytes or natural killer cells in conditioned medium. The scFv-GAS6c disclosed in this application mediates the phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by human monocytes or natural killer cells, regardless of the type of effector cell.

[0117] Furthermore, to verify the effect of the GAS6 fusion protein disclosed in this application on enhancing the phagocytic ability of macrophages or other effector cells after adenovirus infection, in vitro phagocytosis or killing, or other tumor target cell experiments were conducted by co-culturing adenovirus-infected monocytes or other effector cells with N87 or N87-Fluc in the conditioned medium of GAS6 fusion protein. The scFv-GAS6c disclosed in this application mediates the enhanced and promoting effect of human monocytes or natural killer cells infected with adenovirus on targeting N87 or N87-Fluc, or other tumor target cells, which is not limited to the type of effector cells.

[0118] In summary, scFv-GAS6c mediates phagocytic killing of tumor cells based on interaction with TAM RTK, with phagocytes not limited to either the M1 or M2 phenotype of macrophages, nor to effector cells.

[0119] More specifically, when the scFv target of the scFv in the scFv-GAS6c is HER2 (it should be clear that the scFv can be any scFv that targets and kills cells, and is not limited to HER2). Furthermore, the specific preparation steps of the HER2-targeting scFv-GAS6c are as follows:

[0120] Plasmid construction of pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c:

[0121] Gene fragments containing the myc tag, GAS6 signal peptide, anti-HER2 single-chain variable region (scFv), GAS6c, and EPM were sequentially linked, and all sequences were of human origin.

[0122] The amino acid sequence of HER2-scFv-GAS6c-EPM is shown in SEQ ID NO.7; the amino acid sequence of HER2-scFv-GAS6c is shown in SEQ ID NO.8.

[0123] The gene sequence fragments obtained above were ligated into a lentiviral expression vector and synthesized by General Biotech to obtain the plasmid pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c, which can express scFv targeting HER2 and GAS6 interacting with the TAM receptor.

[0124] The plasmid map is shown in Figure 2. The vector pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c uses the EF1a promoter to promote the expression of HER2-scFv-GAS6c.

[0125] Furthermore, the steps for plasmid synthesis are as follows;

[0126] The synthesized plasmid was transiently transfected into 293FT cells, and the supernatant and cells were collected at 24 h, 48 h, and 72 h post-transfection.

[0127] 1. The instant transfection steps are as follows:

[0128] (1) 293FT cells were transferred to 6-well plates (2 mL of complete DMEM), 6 / 7 × 10⁶ cells per well. 5 Cells were transfected the following day.

[0129] (2) 4 μg plasmid in 200 μL PRIME Buffer; vortex.

[0130] (3) Add 8 μL of PRIME to the above mixture, vortex for 10 seconds, and let stand at room temperature for 10 minutes.

[0131] (4) Change the medium 4 hours after transfection (2 mL of complete DMEM).

[0132] (5) Collect the supernatant 24 hours after transfection and replenish the culture medium (2 mL of complete DMEM).

[0133] (6) Collect the supernatant 48 h after transfection and replenish the culture medium (2 mL of complete DMEM).

[0134] (7) Collect the supernatant and cells 72 hours after transfection.

[0135] (8) Using the above construction method, construct a plasmid that can express myc-HER2 scFv-GAS6c.

[0136] 2. Furthermore, the expression of myc-HER2 scFv-GAS6c in cells and supernatant after transient transfection was detected by Western blotting, as follows:

[0137] Cell lysis, protein quantification, SDS-PAGE gel electrophoresis

[0138] (1) Gel preparation: Use One-Step PAGE Gel Fast Preparation Kit (10%).

[0139] (2) Sample preparation: Add 5×Laemmli buffer containing 3% β-mercaptoethanol and 200mM DTT (β-mercaptoethanol and DTT should be added fresh for use) to the cell lysis buffer (5×Laemmli buffer: 0.15M Tris-HCl pH 6.8, 5% SDS, 25% Glycerol and 0.05% Bromophenol blue), in a 95℃ metal bath for 8 min, centrifuge at 12000rpm for 2 min, and use the supernatant for sample loading.

[0140] (3) Loading the sample.

[0141] (4) Electrophoresis: 80V constant voltage electrophoresis.

[0142] (5) Transfer: PVDF membrane activation: Place the membrane in methanol and soak for 5 min. Wet transfer conditions: 80 mA transfer for 180 min.

[0143] (6) Sealing: Place the PVDF membrane in the sealing solution (5% sugar-free soy milk / skimmed milk powder with TBST as solvent) and incubate on a shaker at room temperature for 1-2 hours.

[0144] (7) Primary antibody: Incubate overnight on a shaker at 4°C. Dilute the primary antibody with blocking buffer at a ratio of 1:1000.

[0145] Washing: Incubate on a TBST room temperature shaker for 10 min each time, for a total of 3 times.

[0146] (8) Secondary antibody: Incubate on a shaker at room temperature for 1 hour. Dilute the secondary antibody with blocking buffer at a ratio of 1:2000.

[0147] Washing: Incubate on a TBST room temperature shaker for 10 min each time, for a total of 3 times.

[0148] (9) Exposure.

[0149] Furthermore, the mediated killing effect of the scFv-GAS6c fusion protein on different types of effector cells was verified, namely, the in vitro phagocytosis and killing of N87 cells by human primary macrophages. The results are shown in Figure 4.

[0150] Figure 4 shows the results of HER2-GAS6c fusion protein-mediated phagocytosis of N87 by human primary macrophages in vitro: using normal 293FT complete medium and 293FT complete medium transfected with pCDH-SFFV-copGFP-EF1α-copGFP as negative controls, the HER2-GAS6c fusion protein promoted the phagocytosis of N87 by human primary macrophages compared to the control group. Based on the above experimental data, the phagocytic effect of scFv-GAS6c disclosed in this application on tumor cells is confirmed.

[0151] 1. In vitro phagocytosis of N87 by human primary macrophages

[0152] (1) Count hMDM 3×10 5 Lay the board on a 6-hole plate;

[0153] (2) After 24 hours, hMDM cells were allowed to adhere to the cell wall. Cell Tracker Deep Red was used for adhesion staining at a working concentration of 1 μM. 1 μL of staining solution was added to 106 cells and incubated at 37°C in the dark for 2 hours.

[0154] (3) Discard the supernatant, wash twice with PBS, and then add serum-containing 1640 medium to continue incubation in the dark for later use.

[0155] (4) Count hMDM cells and tumor cells separately, co-culture them at E:T = 1:3, and co-culture them in a 37°C incubator in the dark for 18 hours using the conditioned medium collected after transfection.

[0156] (5) After co-culture, discard the supernatant, wash once with PBS, add 1 ml of trypsin for digestion, neutralize with 2 ml of complete culture medium, centrifuge to collect the cell pellet, and wash once with PBS.

[0157] (6) Add 500 μL PBS to resuspend the sample and perform flow cytometry analysis.

[0158] 2. In vitro killing of N87-Fluc by human primary macrophages:

[0159] (1) Count the hMDM according to the E:T ratio and lay the plates, where E:T = 1:1, 2:1, 3:1;

[0160] (2) After 24 hours, wait for hMDM to adhere to the wall and add 4×10 to each well. 4 N87-Fluc was co-cultured for 18 hours using the conditioned medium collected after transfection.

[0161] (3) After co-culturing, discard the culture medium and add 100 μl of serum-free 1640 culture medium containing D-fluorescein potassium (working concentration 0.3 μg / μl) to each well. Incubate at 37°C in the dark for 10 min and then expose to develop.

[0162] Furthermore, the GAS6 fusion protein described in this application can enhance the in vitro phagocytic ability of adenovirus, as shown in Figure 5.

[0163] Figure 5 shows the results of in vitro phagocytosis of N87-Fluc by RAW264.7 cells infected with adenovirus mediated by the HER2-GAS6c fusion protein. Since the mechanism of action of CAR-M targeting HER2 is the action of ITAM in the intracellular region of the FC receptor, which differs from the mechanism of action of TAM, the synergistic effect of the two leads to stronger phagocytosis and killing of target cells by macrophages. This is especially true given that the transcriptional level of GAS6 binding to the receptor AXL was significantly upregulated by adenovirus-constructed CAR-Ms. Therefore, this study aimed to evaluate whether the secretory HER2-GAS6 fusion protein could further enhance the effect of adenovirus-infected HER2 CAR-M. Experimental results showed that the phagocytic capacity of CAR-M was higher in any culture medium than in the untransduced RAW group and the empty RAW-Ad5F35-mCherry group. Furthermore, the phagocytosis of N87-Fluc mediated by the HER2-GAS6c fusion protein-conditioned medium was more significant than that mediated by the complete culture medium and control culture medium.

[0164] Furthermore, the GAS6 fusion protein disclosed in this application was verified by overexpressing AXL in a stable RAW264.7 strain.

[0165] Furthermore, the specific steps for constructing the stable RAW264.7 strain overexpressing AXL are as follows:

[0166] Constructing a stable RAW264.7 strain overexpressing AXL:

[0167] 1. Plasmid synthesis; pCDH-CMV-AXL-EF1-puro plasmid was synthesized by Universal Biotech (Company);

[0168] 2. Packaging lentiviruses;

[0169] (1) Remove jetPRIME from the 4°C refrigerator, allow it to return to room temperature before use, and mix well before use.

[0170] 293FT cells were transferred to 6-well plates (2 mL of complete DMEM), 6 / 7 × 10⁶ cells per well. 5 Cells, transfected the next day

[0171] (2) The coating plasmid pMD2.G: 0.5 μg, the packaging plasmid pSPAX2: 1.5 μg, and the backbone plasmid: 2 μg were mixed together and added to 200 μL PRIME Buffer; vortexed.

[0172] (3) Add 8 μL of PRIME to the above mixture, vortex for 10 seconds, and let stand at room temperature for 10 minutes.

[0173] (4) Change the medium 4 hours after transfection (2 mL of complete DMEM).

[0174] (5) Change the medium 24 hours after transfection, and then add culture medium (2 mL of complete DMEM).

[0175] (6) Collect the viral supernatant 48h / 72h after transfection. Centrifuge 300g of viral supernatant for 5min, filter through a 0.45μM filter membrane, label it, and aliquot and freeze at -80℃.

[0176] 3. Constructing stable strains

[0177] (1) Seed RAW264.7 cells into 12-well plates one day in advance.

[0178] (2) Add the pre-packaged lentivirus in a 1:1 ratio, i.e., 500 μl of complete DMEM medium and 500 μl of crude virus.

[0179] (3) Change the medium to fresh complete culture medium after 24 hours.

[0180] (4) Observe the fluorescence after 48 hours. Once fluorescence appears, start drug screening.

[0181] 4. Plasmid synthesis

[0182] 5. Packaging Lentiviral

[0183] Furthermore, the experimental steps for stable extracellular phagocytosis and killing of N87 by RAW264.7 overexpressing AXL are as follows:

[0184] (1) Plasmid synthesis; the specific operation steps are the same as above.

[0185] (2) Transfect the synthesized plasmid transiently into 293FT cells, and collect the supernatant and cells at 24h, 48h and 72h after transfection, respectively. The specific operation steps are the same as above.

[0186] (3) WB was used to verify the expression of myc-HER2 scFv-GAS6c in cells and supernatant after transient transfection. The specific operation was the same as above.

[0187] Furthermore, the in vitro phagocytosis experiment of the stable RAW264.7AXL strain with N87 is as follows:

[0188] 1. Count 3 × 10⁻⁶ of RAW264.7AXL. 5 Lay the board on a 6-hole plate.

[0189] 2. After 24 hours, once RAW264.7AXL has adhered to the culture vessel, stain it with CellTracker DeepRed at a working concentration of 1 μM. 6 Add 1 μL of staining solution to the cells and incubate at 37°C in the dark for 2 hours.

[0190] 3. Discard the supernatant, wash twice with PBS, then add serum-containing 1640 medium and continue to culture in the dark for later use.

[0191] 4. Count RAW264.7AXL cells and tumor cells separately, co-culture them at E:T = 1:3, and co-culture them in a 37℃ incubator in the dark for 18 hours using the conditioned medium collected after transfection.

[0192] 5. After co-culture, discard the supernatant, wash once with PBS, add 1 ml of trypsin for digestion, neutralize with 2 ml of complete culture medium, centrifuge to collect the cell pellet, and wash once with PBS.

[0193] 6. Add 500 μL PBS to resuspend the sample and perform flow cytometry analysis.

[0194] Furthermore, the experimental steps for the in vitro killing of N87-Fluc by the stable RAW264.7AXL strain are as follows:

[0195] 1. Lay out RAW264.7AXL according to the E:T ratio, where E:T = 1:1, 2:1, or 3:1.

[0196] 2. After the RAW264.7AXL adheres to the wall, add 4×10 to each hole. 4N87-Fluc was co-cultured for 18 hours using the same conditioned medium collected after transfection.

[0197] 3. After co-culturing, discard the culture medium and add 100 μl of serum-free 1640 medium containing D-fluorescein potassium (working concentration 0.3 μg / μl) to each well. Incubate at 37°C in the dark for 10 min and then expose for development.

[0198] In one specific embodiment, the GAS6 fusion protein is further prepared into an scFv-GAS6c fusion protein antibody drug.

[0199] This application also provides an antibody drug based on the scFv-GAS6c fusion protein. This application describes the use of the scFv-GAS6c fusion protein as an antibody drug for cancer treatment after protein purification using protein purification technology.

[0200] The HER2-scFv-GAS6c-EPM fusion protein was linked using a G4S linker, the amino acid sequence of which is shown in SEQ ID NO.5.

[0201] In vitro experiments were conducted by co-culturing human primary macrophages or other effector cells with N87 or N87-Fluc, or other tumor target cells, and adding purified scFv-GAS6c fusion protein for experimental verification. These experiments demonstrated that the scFv-GAS6c fusion protein mediates the phagocytic killing of N87 or N87-Fluc, or other tumor target cells by human primary macrophages. A mouse orthotopic gastric cancer model was established using N87-Fluc tumor cells, and treatment with scFv-GAS6c fusion protein-based drugs demonstrated that these drugs mediate the phagocytic killing of N87-Fluc, providing a potential strategy for treating gastric cancer or other cancers.

[0202] Furthermore, this application also provides an MSC-based scFv-GAS6c delivery method.

[0203] Kill validation based on MSC delivery of scFv-GAS6c

[0204] According to the embodiments of this application, the nucleotide sequence of TRP1-TA99-scFv-GAS 6c is obtained by artificial synthesis or PCR.

[0205] The plasmid map of TRP1-TA99-scFv-GAS6c is shown in Figure 3.

[0206] The TRP1-scFv-GAS6c-EPM fusion protein, which is linked using the G3S linker, has the amino acid sequence shown in SEQ ID NO.6.

[0207] The nucleic acid sequence of TRP1-TA99-scFv-GAS6c-EPM is shown in SEQ ID NO.9, and the amino acid sequence of TRP1-scFv-GAS6c is shown in SEQ ID NO.10.

[0208] The recombinant plasmid was digested and identified using lentiviral vector pCDH-SFFV-copGFP-EF1a-puro or adenovirus vectors. Results showed that the TRP1-TA99-scFv-GAS6c coding sequence was correctly inserted into the predetermined position of the plasmid. Next, the plasmid was extracted using an endotoxin-free small-volume extract kit from Omega and packaged into lentivirus or adenovirus. Mesenchymal stem cells (MSCs) were infected with the virus. The ability of MSCs infected with TRP1-scFv-GAS6c to phagocytose or kill B16 or B16-Fluc or other tumor cells in vitro was verified, and factor expression after co-culture was detected. In vivo animal experiments were conducted to treat melanoma by intravenously injecting MSCs infected with scFv-GAS6c.

[0209] In summary, due to their superior properties such as promoting tissue repair and regeneration by releasing growth factors and cytokines, facilitating the recruitment of other cells to the site of injury, and regulating the immune system, MSCs, as nutrient cells for delivering scFv-GAS 6c, can be used to treat many other diseases such as osteoarthritis, Crohn's disease, and Parkinson's disease. Furthermore, this application also provides an evaluation method for assessing the tumor-killing effect of stable strains overexpressing AXL.

[0210] According to embodiments of this application, GAS6 exhibits a higher binding affinity for AXL in TAM (TYRO3, AXL, and MERTK) compared to TYRO3 and MERTK. Therefore, this application employs the construction of a stable cell line overexpressing AXL. This stable overexpression cell line can serve as a tool cell for experimental evaluation, replacing monocytes or monocyte-macrophages, among other cells with limited sources.

[0211] First, the CDS sequence of the AXL gene was searched on NCBI. This nucleotide sequence was obtained through artificial synthesis or PCR and inserted into the lentiviral vector pCDH-SFFV-copGFP-EF1a-puro. The recombinant plasmid was then identified by enzyme digestion. Next, the plasmid was extracted using an endotoxin-free mini-prep kit from Omega. The extracted plasmid was then packaged into a stable cell line using jetPRIME. Simultaneously, a lentiviral vector of scFv-GAS6c was constructed. After plasmid extraction, HEK-293T cells were transiently transfected using jetPRIME, and the supernatant was collected as conditioned medium. In vitro experiments were conducted using stable AXL-overexpressing cells co-cultured in conditioned medium to verify that scFv-GAS6c mediates the phagocytosis and killing of B16 or B16-Fluc, or other tumor target cells by the stable AXL-overexpressing cells.

[0212] In summary, this demonstrates that stable cell lines overexpressing AXL can serve as a tool cell to replace monocytes or monocyte-macrophages in verifying their ability to phagocytose and kill tumor cells in vitro.

[0213] Furthermore, the expression of TRP1-TA99-scFv-GAS6c in cells and supernatant after transient transfection was detected by Western blotting, as described above, and the results are shown in Figure 6.

[0214] Figure 6 shows the expression of the TRP1-TA99-scFv-GAS6c fusion protein in cells and supernatant after transient transfection:

[0215] The HER2-GAS6c fusion protein can link one end to HER2-positive tumor cells and the other end to macrophages, thus acting as a supernatant addition and bridging the gap. This allows macrophages to target and bind to HER2-positive tumor cells via this dual-ended linker, thereby eliminating the target cells. Western blotting analysis of HER2-GAS6c protein expression in cells and supernatants after transient transfection with pCDH-SFFV-copGFP-EF1α-HER2-GAS6c to 293FT confirmed normal HER2-GAS6 secretion.

[0216] More specifically, the ability of RAW264.7 to phagocytose B16 in vitro was verified using TRP1-TA99-scFv-GAS6c conditioned medium, and the results are shown in Figure 7.

[0217] Figure 7 shows the results of RAW264.7 phagocytosis of B16 mediated by the TRP1-TA99-GAS6c fusion protein in vitro:

[0218] Using 293FT complete medium and 293FT complete medium transfected with pCDH-SFFV-copGFP-EF1α-copGFP as negative controls, and pCDH-SFFV-copGFP-EF1α-GAS6 conditioned medium expressing full-length GAS6 as a positive control, the TRP1-TA99-GAS6c fusion protein significantly promoted the phagocytosis of B16 by RAW264.7 cells compared to the control group. Based on the above experimental data, the phagocytic effect of scFv-GAS6c disclosed in this application on tumor cells is confirmed.

[0219] The embodiments described above merely illustrate several implementation methods of this application to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

A GAS6 fusion protein characterized in that, From the N-terminus to the C-terminus, it includes antigen-specific binding protein, linker, and GAS6c; The amino acid sequence of GAS6c is shown in SEQ ID NO.

1. The GAS6 fusion protein according to claim 1, characterized in that, The C-terminus of the GAS6 fusion protein also includes a fragment encoded by the EPM gene, as shown in SEQ ID NO.

2. The GAS6 fusion protein according to claim 1, characterized in that, The amino acid sequence of the scFv is shown in SEQ ID NO.3 or SEQ ID NO.

4. The GAS6 fusion protein according to claim 1, characterized in that, The N-terminus of the GAS6 fusion protein also includes a signal peptide as shown in SEQ ID NO.11; The signal peptide includes one or more of GAS6, CSF2RA, and hCD8A. The GAS6 fusion protein according to claim 1, characterized in that, The linker includes one or more flexible linkers. The GAS6 fusion protein according to claim 5, characterized in that, The amino acid sequence of the linker is shown in SEQ ID NO.5 or SEQ ID NO.

6. The GAS6 fusion protein according to any one of claims 1 to 6, characterized in that, The antigen-specific binding protein includes one or more of single-chain antibodies, ligands, and antibodies modified to bind. The GAS6 fusion protein according to any one of claims 1 to 6, characterized in that, Its amino acid sequence is shown in SEQ ID NO.7 or SEQ ID NO.

8. The GAS6 fusion protein according to any one of claims 1 to 6, characterized in that, Its amino acid sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10. The GAS6 fusion protein according to any one of claims 1 to 6, characterized in that, The GAS6 fusion protein was delivered via MSC. A nucleic acid molecule, characterized in that, It includes the GAS6 fusion protein as described in any one of claims 1 to 9. A recombinant vector, characterized in that, It comprises the nucleic acid molecule of claim 11; The recombinant vector is either a eukaryotic expression vector or a prokaryotic expression vector. A recombinant cell, characterized in that, The recombinant cells express the GAS6 fusion protein of any one of claims 1 to 9, or contain the nucleic acid molecule of claim 11 or the recombinant vector of claim 12; The recombinant cells are eukaryotic cells or prokaryotic cells. A method for preparing a GAS6 fusion protein, characterized by, include: The recombinant cells of claim 13 are cultured, and the GAS6 fusion protein is isolated from the resulting culture. A pharmaceutical composition, characterized by The pharmaceutical composition contains the GAS6 fusion protein as described in any one of claims 1 to 9, and pharmaceutically acceptable excipients. The pharmaceutical composition according to claim 15, characterized in that, The pharmaceutical composition also includes immune cells. The pharmaceutical composition according to claim 16, characterized in that, The immune cells include one or more of monocytes or macrophages, dendritic cells, natural killer cells, or natural killer T cells; The macrophages mentioned therein include M1 macrophages or M2 macrophages. A method of treating a disease in a subject, characterized by, include: The pharmaceutical composition of claims 15-17 is administered to a subject in a therapeutically effective amount.

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